The proposed FIRCA project will be carried out primarily in the Institute of Analytical Chemistry in Prague, Czech Republic, in collaboration with Dr. Jiri Dedina as an extension of NIH Grant 1 R01 ES010845. The metabolism of inorganic arsenic (iAs) in humans involves reduction and methylation reactions that produce methylated trivalent and pentavalent arsenicals. The liver is likely the major site for methylation of iAs in human body. The methylated trivalent arsenicals, unlike their pentavalent counterparts, are more potent than iAs as cytotoxins, genotoxins, enzyme inhibitors, and as modulators of major signal transduction pathways in human cells. Thus, formation of these species in the methylation pathway for iAs is often regarded as a mechanism for activation of this metalloid as a toxin and carcinogen. The interindividual variations in the capacity of the liver to methylate iAs may underlie differences in the susceptibility of individuals to toxic and carcinogenic effects associated with acute and chronic exposures to iAs. The proposed FIRCA project is an extension of the parent NIH-funded project that examines qualitative and quantitative aspects of the interindividual variations in the metabolism of iAs in human hepatic tissue and in primary human hepatocytes obtained from a significant number of donors. In the parent project, trivalent and pentavalent metabolites of iAs are analyzed in tissues and in hepatocyte cultures exposed to various concentrations of iAs, using the oxidation state specific hydride-generation (HG)-atomic absorption spectrometry (AAS). This method is uniquely suited for the speciation analysis of arsenic in complex biological matrices. However, it lacks sensitivity that is required for analysis of small amounts of metabolites generated under conditions of low iAs exposures. The goal of the proposed FIRCA project is to optimize the HG methodology and instrumentation to increase sensitivity of the arsenic speciation analyses by AAS or by atomic fluorescence spectrometry. The optimized HG systems will then be used in the parent project for analysis of metabolites produced by human hepatocytes exposed to low concentrations of iAs that are consistent with chronic environmental exposures. Importantly, the ultimate goal of the proposed research is to further improve the capacity of widely-used HG-based analytical techniques to analyze iAs metabolites, particularly the toxic methylated trivalent intermediates, in body fluids and tissues of individuals exposed to low concentrations of iAs from the environment.